The Nef and Vpu accessory proteins of HIV-1 - Primate immunodeficiency viruses target helper T-cells and macrophages/monocytes through binding of the viral envelope glycoprotein to a combination of CD4 and a chemokine receptor (CCR4 or CXCR5) on the surface of the host cells. Strikingly, infection results in rapid and sustained downregulation of CD4 and, to a lesser extent, the chemokine receptors. Downregulation of these viral co-receptors prevents superinfection, promotes virion release and interferes with the immune response, leading to the establishment of a robust infection. CD4 downregulation is so important to the life cycle of human immunodeficiency virus-1 (HIV-1) that two accessory proteins, Nef and Vpu, encoded in the viral genome are devoted to this task. Indeed, Nef and Vpu are critical for the progression from infection to AIDS, a fact that is best illustrated by the existence of long-term non-progressors that are infected with HIV-1 strains bearing inactivating mutations in the genes encoding these proteins. Therefore, pharmacologic or biologic perturbation of Nef and/or Vpu has the potential to prevent the pathogenic effects of HIV-1. To date, however, this potential has not been realized mainly because Nef and Vpu have no enzymatic activity and their mechanisms of action are insufficiently understood. Structural basis for CD4 downregulation by the Nef protein of HIV-1 - In previous work, we made substantial progress towards elucidating the mechanism of CD4 downregulation by Nef. We found that Nef connects surface CD4 to both the endocytic and lysosomal targeting machineries, leading to efficient and sustained removal of CD4 from the host cells early during infection. We discovered that the role of Nef in CD4 internalization involves an interaction with the AP-2 clathrin adaptor. A dileucine motif and a diacidic motif in a C-terminal loop of Nef were found to be essential for interaction with a site on the AP-2 alpha-sigma2 hemicomplex and for CD4 downregulation, but the structural details of these interactions were not known. In collaboration with James Hurley (NIDDK, now at UC Berkeley), we solved the crystal structure of Nef bound to the alpha and sigma2 subunits of AP-2. The structure revealed that the Nef dileucine motif directly interacts with a binding site for host dileucine-signal containing cargo proteins on alpha-sigma2. The Nef diacidic motif, on the other hand, does not directly interact with AP-2, but stabilizes a binding-competent conformation of the central loop. In addition, the structure showed that the Nef core is involved in direct contacts with alpha-sigma2 while also serving as a scaffold to position the central loop. Mutagenesis in conjunction with AP-2 binding and CD4 downregulation analyses confirmed the importance of the residues identified in the crystal structure. The new interfaces revealed by these analyses are not known to be used by any host cell transmembrane proteins and may therefore be specific for Nef. If so, they may represent an Achilles heel that could be exploited for the development of novel anti-Nef agents. The host cell protein Alix/AIP1 is required for targeting of internalized CD4 to the multivesicular body pathway by Nef - Subsequent to induction of CD4 internalization by an AP-2/clathrin pathway, Nef promotes delivery of internalized CD4 to the multivesicular body pathway (MVB) for eventual degradation in lysosomes. In previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. In collaboration with Luis daSilva (University of Sao Paulo, Brazil) we recently found that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We showed that Nef interacts with both the Bro1 and Vdomains of Alix. Depletion of Alix or overexpression of the Alix V domain impaired lysosomal degradation of CD4 induced by Nef. In contrast, V-domain overexpression did not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical, ubiquitination-dependent MVB pathway. We also showed that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, these results indicated that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.